Deletion of the epidermal water/glycerol transporter aquaporin-3 (AQP3) in mice reduced superficial skin conductance by ϳ2-fold (Ma, T., Hara, M., Sougrat, R., Verbavatz, J. M., and Verkman, A. S. (2002) J. Biol. Chem. 277, 17147-17153), suggesting defective stratum corneum (SC) hydration. Here, we demonstrate significant impairment of skin hydration, elasticity, barrier recovery, and wound healing in AQP3 null mice in a hairless (SKH1) genetic background and investigate the cause of the functional defects by analysis of SC morphology and composition. Utilizing a novel 3 H 2 O distribution method, SC water content was reduced by ϳ50% in AQP3 null mice. Skin elasticity measured by cutometry was significantly reduced in AQP3 null mice with ϳ50% reductions in elasticity parameters Uf, Ue, and Ur. Although basal skin barrier function was not impaired, AQP3 deletion produced an ϳ2-fold delay in recovery of barrier function as measured by transepidermal water loss after tape stripping. Another biosynthetic skin function, wound healing, was also ϳ2-fold delayed by AQP3 deletion. By electron microscopy AQP3 deletion did not affect the structure of the unperturbed SC. The SC content of ions (Na ؉ , K ؉ , Ca 2؉ , Mg 2؉ ) and small solutes (urea, lactic acid, glucose) was not affected by AQP3 deletion nor was the absolute amount or profile of lipids and free amino acids. However, AQP3 deletion produced significant reductions in glycerol content in SC and epidermis (in nmol/g protein: 5.5 ؎ 0.4 versus 2.3 ؎ 0.7 in SC; 0.037 ؎ 0.007 versus 0.022 ؎ 0.005 in epidermis) but not in dermis or blood. These results establish hydration, mechanical, and biosynthetic defects in skin of AQP3-deficient mice. The selective reduction in epidermal and SC glycerol content in AQP3 null mice may account for these defects, providing the first functional evidence for physiologically important glycerol transport by an aquaporin.Hydration of the stratum corneum (SC), 1 the non-viable outermost layer of skin, is an important determinant of skin appearance, metabolism, mechanical properties, and barrier function (1-3). Water is continuously exchanged among the SC, the underlying viable epidermis, and the external atmosphere. SC water content depends on external humidity, the capacity of the epidermis to replace evaporative water losses, and the intrinsic SC "water holding capacity" (4). The determinants of SC water holding capacity are thought to include SC structure and composition, particularly the content of small molecule osmolytes or "humectants" such as free amino acids (5, 6). Decreased SC water content is found is a number of common skin diseases such as atopic dermatitis (7), eczema (8), psoriasis (9), senile xerosis (10), and hereditary ichthyosis (11).The water/glycerol transporting protein aquaporin-3 (AQP3) is expressed in the basal (innermost) layer of keratinocytes in mammalian epidermis as originally shown in rat skin (12) and then in human (13) and mouse (14) skin. AQP3 facilitates the transmembrane transport of water in response t...